Keywords PDZ proteins · Proximal tubule · Reabsorption
In the kidneys a large part of filtered solutes is reab-sorbed by specific transporters localized in the microvilli of the proximal tubular cells (PT cells). Depending on the body needs, the rates of reabsorption of certain sol-utes are adjusted mostly by hormonal control mecha-nisms. Most transporters are distributed along the whole microvilli (approx. 1 µm of length) and in the case of regulated transporters, they can also be detected in the intermicrovillar clefts – the sites of internalization – as-sociated with clathrin-coated pits/vesicles and in endoso-mal structures contained in the subapical compartment. From the latter, internalized transporters may recycle back to the apical membrane or may be routed to the ly-sosomes. This arrangement of regulated and non-regulat-ed transporters suggests that probably all transporters in-teract in some way with other proteins. Such inin-teractions are thought to be involved in: (1) the targeting of trans-porters to the apical membrane, (2) keeping microvillar transporters in place, (3) recruiting elements of the sig-nalling cascades involved in regulating transport pro-cesses and (4) the processing of internalized transporters in the subapical compartment.
Very recently, we have begun to learn how supramo-lecular structures such as synapses are organized in terms of static and dynamic protein–protein interactions [3, 4, 13]. Proteins containing PDZ domains (name de-rived from the postsynaptic protein PSD95, dlg-A from Drosophila and the tight junction protein ZO-1) have emerged as important elements in organizing such mem-brane complexes. Several PDZ proteins have been iden-tified that are expressed in epithelial cells and localized at the apical or the basolateral membrane. In this short commentary I shall restrict discussion to PDZ proteins
localized in the brush borders of PT cells and speculate about their possible functions regarding the sorting/posi-tioning and regulation of solute transporters.
Microvillar PDZ complexes
To date, three PDZ proteins have been described in the brush borders (microvilli and subapical compartment) of PT cells: NHERF-1 (also named EBP50) and proteins named NaPi-Cap1/2 (also known as PDKZ1, Cap70 or CLAMP).
NHERF-1
Originally NHERF-1 (encompassing two PDZ domains in tandem) was identified as a regulatory factor of the Na/H-exchanger NHE-3 and since has been established to interact with NHE-3 at or close to PDZ domain 2 [14]. In addition, evidence was obtained that NHERF-1 (via PDZ domain 1) also interacts with the C-terminus of the NaPi-lla protein [5] and with the adrenergic receptor β2 [6]. Furthermore, EPI64, a protein containing a TBC/rab-GAP domain implicated to play a role in vesicular mem-brane traffic, was recently identified to bind to the PDZ domain 1 of NHERF-1 as well [11]. Although EPI64 is abundant in kidney tissue its cellular localization re-mains to be determined.
On the other hand, NHERF-1 interacts with ezrin, a member of the ERM family of actin binding proteins. Ezrin itself interacts with actin filaments and additional-ly provides an anchoring site for protein kinase A.
NaPi-Cap proteins
Based on a yeast two-hybrid screen, two PDZ proteins were identified which interact with the C-terminus of the Na/Pi-cotransporter type lla: NaPi-Cap1 and NaPi-Cap2 (85% homologous to NaPi-Cap1) [5]. Both proteins
en-J. Biber (
✉
)Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland e-mail: JuergBiber@access.unizh.ch
Fax: +41-1-6355715
Pflügers Arch - Eur J Physiol (2001) 443:3–5 DOI 10.1007/s004240100721
F R O N T I E R S I N P H Y S I O L O G Y
J. Biber
Emerging roles of transporter-PDZ complexes
in renal proximal tubular reabsorption
Accepted: 21 August 2001 / Published online: 25 September 2001 © Springer-Verlag 2001
compass four PDZ domains in tandem. Immunohisto-chemistry revealed that, in the kidney, these two proteins are only localized in proximal tubules: NaPi-Cap1 was predominantly observed in the microvilli, whereas NaPi-Cap2 was preferentially observed in the subapical re-gion.
The interaction of NaPi-lla with the NaPi-Cap1/2 pro-teins was mapped to PDZ domain 3 and occurs predomi-nantly via the last three amino acids (TRL). Other proxi-mal tubular apically expressed proteins, MRP2 and a small, cancer-associated, protein named MAP17, were reported to interact with NaPi-Cap1 as well [8].
Functional consequences
of PDZ-transporter interactions
Best described examples of regulated proximal tubular membrane transport proteins are the Na/H-exchanger NHE-3 [9] and the Na/phosphate cotransporter NaPi-lla [10]. The following roles (experimentally established and speculated) of PDZ interactions with these transport-ers in the brush bordtransport-ers of PT cells can be envisaged.
Apical membrane targeting/positioning
Roles of PDZ interactions have emerged more and more in the targeting and correct positioning of membrane proteins. In the case of the cotransporter NaPi-lla, it was shown that apical expression of the transporter in OK-cells could be disturbed by truncating the last three C-terminal amino acids and thereby preventing the interac-tion with PDZ domain 3 of NaPi-Cap1 [7]. It has yet to be determined if this observation is due to a defect in the targeting machinery as such or a defect in the final posi-tioning.
Control of transport rates
Both transporters, NHE-3 and NaPi-lla, are regulated by parathyroid hormone (PTH), among others. PTH inhibits these transporters in different ways: inactivation of the NHE-3 exchanger initially occurs through phosphoryla-tion “in situ” followed by internalizaphosphoryla-tion [9], whereas the NaPi-lla cotransporter undergoes immediate internaliza-tion upon PTH receptor stimulainternaliza-tion [10]. Moreover, in-ternalized NHE-3 transporters are recycled back from the subapical compartment to the apical membrane, whereas internalized NaPi-lla cotransporters are routed to the lysosomes. How could PDZ interactions partici-pate in such regulatory events?
a. By anchoring components of signalling pathways? PTH stimulates PKA activity, and PKA-mediated phosphorylation inhibits NHE-3 transport activity. It has been established that the phosphorylation of NHE-3 depends on the interaction with the
NHERF-1/ezrin complex, which serves as an anchor site for PKA. Interestingly, phosphorylation (probably via a G-protein-coupled receptor kinase 6A) of NHERF-1 was demonstrated as well; however, the interaction of NHERF-1 with NHE-3 does not seem to depend on such phosphorylation and appears to be constitutive. In contrast, the interaction of NHERF-1 with the β2-receptor appears to be more dynamic and depends on receptor stimulation [2].
It is not yet known whether the NHERF-1/ezrin/PK-A complex is necessary for regulation of the NaPi-lla transporter, or whether NaPi-Cap1 also provides an-choring sites for regulatory elements (PKA or PKC). b. By directly interacting with transporters? Although
possibly not directly relevant to the regulation of NHE-3 and NaPi-lla, studies of the interaction of CFTR with NaPi-Cap1 and NHERF-1 provide evi-dence for a new phenomenon of how PDZ interac-tions may modulate channel (transporter) activities [1]. Such activation may occur through the recruit-ment of regulatory subunits or the stabilization of oli-gomeric forms of channels or transporters. Note, how-ever, that the presence of CFTR in the apical mem-brane of PT cells remains somewhat controversial. c. By participating in the migration of transporters along
the microvilli? Internalization of NaPi-lla and NHE-3 occurs in intermicrovillar clefts. How do transporters reach this site? It is currently not clear whether trans-porters constantly move from the clefts to the tip of microvilli and back again, and are then internalized at the clefts upon a signalling event, or if movement down to the clefts is provoked by a specific signal. In any case, both models suggest that interactions of these transporters with PDZ proteins may not be of a static nature but may exist in an “on-off” mode. d. By sorting internalized transporters? Both
transport-ers, NHE-3 and NaPi-lla, are internalized at intermi-crovillar clefts via clathrin-coated pits/vesicles. Inter-nalized transporters are either recycled back to the apical membrane (NHE-3) or routed to the lysosomes (NaPi-lla). This implies that NHE-3 and NaPi-lla con-tained in endosomal structures of the subapical com-partment are handled differently. Interestingly, the PDZ domain 4 protein NaPi-Cap2 was found predom-inantly in the subapical compartment and was found to interact with NaPi-lla, but not with NHE-3. This suggests that NaPi-Cap2 may play a role in the sort-ing and routsort-ing of internalized NaPi-lla cotransport-ers. A possible role of NHERF-1 in the recycling of NHE-3 can be envisaged, analogous to the recycling of the β2-receptor for which the importance of NHERF-1 has been demonstrated [12].
How complex will it be?
To date, in brush borders of PT cells, only a few mem-brane protein-PDZ interactions have been described (Fig. 1). Certainly there will be many more, as indicated
by the still empty sites of the NaPi-Cap1/2 proteins. Most of these interactions are via a class I PDZ binding motif (S/T-X-L) but we have to assume that interactions based on class II PDZ binding motifs (φ–X–φ) also occur; the corresponding PDZ proteins remain to be identified.
Current results suggest that certain proteins interact exclusively with one PDZ domain while others may inter-act with several PDZ domains such as the NaPi-lla co-transporter, which interacts with both NHERF-1 and NaPi-Cap1. Clearly, the relative affinities of such multi-ple interactions remain to be determined. But importantly, these findings also raise the question about possible mod-ulations of PDZ interactions. Controlled on-off reactions may be necessary, such as in the case of the regulation of the NaPi-lla and the NHE-3 transporter. On-off reactions may be necessary (1) to allow a controlled movement of these transporters along the microvilli or (2) to control in-ternalization at the intermicriovillar clefts.
Taken together we shall be confronted with an orches-trated assembly of static and dynamic interactions of PDZ domains with membrane proteins (transporters, re-ceptors) and cytosolic proteins (cytoskeletal and signal-ling elements) in the microvillus of PT cells. To date, most PDZ interactions have been described by in vitro
approaches and by studies performed on cell cultures. It will be a formidable task to unravel the precise physio-logical and pathophysiophysio-logical roles of the known and as yet unidentified PDZ interactions in the microvilli of PT cells using all available techniques.
Acknowledgements The author is grateful for all the
experimen-tal and theoretical help provided by Heini Murer, Nati Hernando, Serge Gisler, Sandra Pribanic and Ian Forster.
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Fig. 1 Schematic arrangement of described PDZ interactions in
the brush borders (microvilli and subapical compartment) of prox-imal tubular cells. (EV Endosomal vesicle.) Interactions that re-main to be identified are indicated by a clear three-quarter circle
